Two earlier studies-using different criteria to characterize intracellular-pH (pHi) physiology-each identified two functional classes of neurons. In the first study, the initial pHi in a CO2/HCO3-free (i.e., HEPES- buffered) solution defines two populations of freshly dissociated pyramidal hippocampal (HC) neurons, a relatively low-pHi neuronal population (80%) and a relatively high-pHi neuronal population (20%). The second study comparing the response to acid/ base disturbances in HC and MR neurons saw similar results during respiratory acidosis, respiratory alkalosis, and metabolic alkalosis . The second criterion is the sensitivity to metabolic acidosis (MAc), a decrease in intracellular pH (pHo) caused by a decrease in [HCO3-] at fixed [CO2]. About 20% of cultured HC neurons are MAc-sensitive, exhibiting a large pHi decrease in response to MAc, whereas ~80% are MAc-resistant. In the medullary raphe (MR),15% of neurons were MAc-resistant and 85% were MAc-sensitive. An exciting preliminary result is that a second episode of MAc causes MAc-sensitive HC neurons to become MAc-resistant-perhaps the first example of history-dependent modulation of acid-base transport. Differences in CO2/HCO3-dependent transporter expression or regulation may explain differences in pHi regulation and MAc sensitivity. MR neurons are chemosensitive and altering the acid- base regulation through MAc exposure may affect their respiratory chemosensitivity and/or firing rate. Aim 1. To define the molecular basis of MAc sensitivity vs. MAc resistance using pH sensitive electrodes, immunocytochemistry, single cell PCR, KOs, and Western Blot. (A) Do MR neurons (like HC neurons) exist in high-pHi vs. low-pHi states, and are the high-pHi neurons identical to the MAc-sensitive ones? (B) Does MAc sensitivity vs. resistance correlate with the presence of specific SLC4 transporters, RPTP gamma or beta, or a specific neurotransmitter? (C) Does the knockout of specific SLC4 transporters, RPTP gamma, or RPTP beta shift the ratio of MAc-sensitive vs. resistant (or high- vs. low-pHi) neurons? Aim 2. To determine if a second MAc exposure (as in HC neurons) shifts MAc- sensitive MR neurons toward MAc resistance. Aim 3. To determine if pH-chemosensitivity in MR neurons correlates with pHi physiology. I will use patch clamp techniques. (A) Does the ability of MR neurons to alter firing rate in response to MAc correlate with MAc sensitivity vs. resistance, SLC4 profile, RPTP expression, or transmitter content. (B) Must pHi change in order for an MR neuron to alter firing rate? The proposed work will provide a valuable training experience as well as important new insights into respiratory control and could have important implications for several respiratory diseases, including sleep apnea, SIDS.